Transcript MRC Unit The Gambia PowerPoint Template

DNA methylation as an epigenetic
marker in HIV-2 disease in West
Africa?
Alberta Davis
MRC Laboratories, Gambia
18th January 2013
MRC Unit, The Gambia
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HIV pathogenicity and clinical outcomes
• AIDS: causal agents; retroviruses
» HIV-1
» HIV-2
• HIV-1 “elite” controllers, long term non progressors (LTNPs),
exposed uninfected. (Saksena et al 2007)
• HIV-2 infection is less progressive (low VL, low transmission,
slow decline of CD4 T cells, prolonged survival).
• 20% of HIV-2 infected individuals exhibit high VL and a
clinical presentation indistinguishable from AIDS in HIV-1.
(Esbjörnsson et al, 2012)
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Determinants of progression to AIDS
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Host determinants
• CCR5 receptor:
• Viral entry into CD4 T cells.
• mutation confers resistance to infection with HIV.
• Variable CCR5 expression in HIV-1 is associated with matrices
of infection and pathogenesis.
• Low and protective levels of CCR5 on CD4+ T cells is exhibited
in HIV-2 infections (Shea et al 2004).
• IL-2 –CCR5 are coregulated genes and CCR5 can influence signaling
events during T cell activation (Camargo et al 2009).
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Epigenetics and its regulation of genes
• Epigenetic mechanisms:
 Heritable but reversible without change in DNA sequence
 DNA methylation in CpG dinucleotide = gene repression
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hypothesis
“ The DNA methylation status of regulatory regions of CCR5 and
IL-2 serve as a determinant of differential HIV-2 pathogenicity”
• Objective
» To determine the methylation status at CCR5 cisregulatory CpG sites and IL-2 gene loci in progressive
and non progressive infections
» Evaluate the frequencies of CCR5 genetic variants
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Methods
• HIV-2 rich cohorts – Guinea Bissau and Gambia.
• Interrogated patient database based on previous studies of
HIV-2 non progression (Berry et al 2002, Schim van der Loeff et al 2010).
• Categorised samples into various groups based on CD4 and
Viral load
• Progressor: CD4 <200 cells/ul and VL > 10,000 copies/ml
• Non progressor: CD4 >500 cells/ul and VL < 100 copies/ml
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Overview of patient profiles
Demographic and virologic characteristics of 36 HIV-2 subjects
n
%
Characteristic
Categories/units
Age: Median(range)
years
45 (21-73)
CD4: Median(range)
counts
573 ( 10 - 2720)
Viral load: Median(range)
counts
22146 ( 100 - 607000)
Sex
Male
Female
15
21
41.7
58.3
Ethnicity
Mandinka
Manjago
Other
13
9
14
36.1
25
38.8
CD4 counts
Low (< 200 cells/ul)
High (> 500 cell/ul)
15
21
41.7
58.3
Viral load counts
Low (< 100 copies/ml)
High (> 10,000 copies/ml)
15
21
41.7
58.3
CD4/VL
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H/H
H/L
L/H
L/L
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9
25
12
33.3
12
33.3
3 health across
8.3the developing world
to save lives and improve
Specific CpG in CCR5 cis regulatory sites
Bisulphite modification and pyrosequencing (CCR5 and IL-2)
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Association between DNA methylation and CD4
CD4 > 500 (n = 21)
CD4 > 200 (n = 15)
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Correlation CD4 and methylation of CCR5
and IL-2
Methylation against CD4 at IL-2
n=36, rho=0.501 p value=0.002
n=36, rho=-0.454 p value=0.005
50
5
30
10
40
15
20
Methylation
60
25
70
30
Methylation against CD4 at Distal Window
2
4
6
Log(CD4)
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8
2
4
6
8
Log(CD4)
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Progressor (L/H) and non progressor (H/L)
phenotypes show different methylation patterns
L/H (n = 12)
H/L (n = 12)
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Conclusion
• The CD4 count and VL load could be influenced by methylation
levels.
• Viral control in non progressors is being achieved by low CCR5
expression and maintenance of CD4+ T cells which produce
IL-2.
• Progressors are less methylated at CCR5 regulatory regions
but more methylated at the IL-2 locus than non progressors.
• No significant difference was found at CCR5 promoter 2
possibly because Pr2 is active only upon T cell activation
MRC Unit, The Gambia
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Recruitment of patient and blood sampling in
Guinea Bissau
Duration in the cohort (1989, 1997, 2003, 2006)
• HIV-2 non progressor,HIV-2 progressor,HIV-1
asymptomatics, Healthy controls.
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•
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CCR5 and IL-2 DNA methylation
CCR5 and IL-2 mRNA expression levels
CCR5 allelic discrimination
Immunophenotyping by flow cytometry
Multiplex cytokine analysis by Bioplex-cytokine analysis
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Acknowledgements
UTHSCSA
MRC Unit
Prof Sunil K. Ahuja
Dr He Weijing
Dr Assan Jaye
Komathy Jayasekar
Una Aluyen
Shivali Chag
MRC Unit, The Gambia
Pa Saidou Chaw, MD
Dr Alfred Ngwa
Ramou-Sarge Njie
Gilleh Thomas
James Jafali
Leading scientific research to save lives and improve health across the developing world
THANK YOU!
MRC Unit, The Gambia
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